Abstract
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.
Highlights
Cryptosporidium is a genus of protozoan pathogens belonging to the Phylum Apicomplexa that infect humans and/or animals
We have observed that most C. parvum cultured in 24- or 48-well plates may complete the second generation of merogony and a large number of free merozoites may be released into the culture medium at 48 hpi
We generated standard curves from cells infected with various numbers of parasites. We considered that this approach was better than the traditional method using serially diluted RNA templates, because standard curves derived from various numbers of infected parasites would correct systematic errors from infection, sample preparation to qRT-PCR procedures, rather than to only correct the errors introduced at the qRTPCR step
Summary
Cryptosporidium is a genus of protozoan pathogens belonging to the Phylum Apicomplexa that infect humans and/or animals. C. parvum and C. hominis are the two major species causing water-borne and food-borne diarrheal illness in humans (Baldursson and Karanis, 2011; Budu-Amoako et al, 2011). These two and some other species, including C. meleagridis, C. muris, C. canis, and C. felis, cause opportunistic infections in AIDS patients (AIDS-OIs) that can be chronic and fatal (Thompson et al, 2005; Feasey et al, 2011; O’connor et al, 2011; Shirley et al, 2012). Only a single drug, nitazoxanide (NTZ) is approved by the U.S Food and Drug Administration (FDA) to treat
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